2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

other: stability

Qualifier:

no guideline available

Principles of method if other than guideline:

The purpose of this study was to determine the relative stability of test item in human plasma as compared to other species in order to determine the suitability of the rat as a species for toxicology studies.

GLP compliance:

no

Specific details on test material used for the study:

- Storage condition of test material: Stored at approximately 25 °C away from direct sunlight until the day of the experiment and/or analysis

Radiolabelling:

no

The test item (750 ng/mL) was incubated with dog, human, minipig, monkey, rabbit, or rat plasma at 37°Cfor 24 hours. Peak area of the test item and S8551 (internal standard) as well as the ratio of peak area of test item/S8551, and normalized data in the various species are summarized in Table 1.

The test item was rapidly hydrolyzed in rat plasma with only 3.8% remaining at the 15 minute time point. In contrast, the test item was very stable in human, dog, and pig plasma with 90.0%, 110.4%, and 91.7%, respectively, remaining at the 24 hour time point. The test item was less stable in either monkey or rabbit plasma with 36.7% and 20.1%, respectively, remaining at the 24 hour time point. The results indicate that the plasma stability of the test item in humans is most similar to either dog or Gottingen pig.

Conclusions:

The test item was rapidly hydrolyzed in rat plasma with only 3.8% remaining at the 15 minute time point. In contrast, the test item was very stable in human, dog, and pig plasma with 90.0%, 110.4%, and 91.7%, respectively, remaining at the 24 hour time point. The test item was less stable in either monkey or rabbit plasma with 36.7% and 20.1%, respectively, remaining at the 24 hour time point. The results indicate that the plasma stability of the test item in humans is most similar to either dog or Gottingen pig.

Executive summary:

A study was conducted to determine the relative stability of the test item in human plasma as compared to other species in order to assess the suitability of the rat as a species for toxicology studies.

The test item (750 ng/mL) was incubated with human, rat, dog, rabbit, monkey, or Gottingen pig plasma at 37 °C for 24 hours. Duplicate aliquots (50 µL) were taken at 0, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, and 24 hours and diluted into acetonitrile (150 µL) containing an internal standard (S8551). The samples were centrifuged to separate the precipitated proteins from the supernatant. The supernatant was analyzed by LC-MS/MS using an API 3200 QTrap equipped with an Agilent 1100 HPLC system in order to determine the percent of the test item remaining at the various time points. All data were normalized by ratio of peak area (test item/S8551) at the 0 hour time point.

 

The test item was rapidly hydrolyzed in rat plasma with only 3.8% remaining at the 15 minute time point. In contrast, the test item was very stable in human, dog, and pig plasma with 90.0%, 110.4%, and 91.7%, respectively, remaining at the 24 hour time point. The test item was less stable in either monkey or rabbit plasma with 36.7% and 20.1%, respectively, remaining at the 24 hour time point. The results indicate that the plasma stability of the test item in humans is most similar to either dog or Gottingen pig.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

absorption

Qualifier:

no guideline available

Principles of method if other than guideline:

The objective of this study was to determine the pharmacokinetic parameters of the test item and its hydrolysis metabolites (S2225 and S5353) in plasma following single oral administration as well as single intravenous administration in male beagle dogs.

GLP compliance:

no

Radiolabelling:

no

Species:

dog

Strain:

Beagle

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Animal testing was conducted at Charles River Laboratories (Study Number 20033875). All animals were fasted overnight prior to treatment (total fasting time did not exceed 12 hours). On test day, dogs were weighed and randomly assigned to study groups.

Route of administration:

other: gelatin capsules by oral gavage and intravenous administration

Vehicle:

other: gelatin capsule (oral) and PEG 400 solution (intravenous)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
All formulations were prepared by Charles River (Study Number 20033875). For each oral dose, the capsules (1 gelatin capsule per animal) were prepared by weight based on individual body weights obtained on the day of dosing. For each IV dose, the required amount of the test item was dissolved in Polyethylene Glycol 400 (PEG 400) to make a 5 mg/mL solution. Dose formulation samples were shipped on dry ice to the Analytical Department at the test facility for analysis on October 1, 2012 and were stored at approximately -20 °C until analysis.

For IV dosing,the test item was administered as a bolus intravenous injection into a peripheral vein by way of a temporary percutaneous catheter. The dosing catheter was flushed with 3 mL of saline immediately after dosing prior to removal. Dose capsules were administered manually. Following each capsule dose, 10-20 mL of water was offered to each animal to assist in swallowing the capsule.

Duration and frequency of treatment / exposure:

Single dose

Dose / conc.:

1 mg/kg bw/day

Remarks:

intravenous

Dose / conc.:

10 mg/kg bw/day

Remarks:

oral (gelatin capsule)

Dose / conc.:

30 mg/kg bw/day

Remarks:

oral (gelatin capsule)

Dose / conc.:

100 mg/kg bw/day

Remarks:

oral (gelatin capsule)

No. of animals per sex per dose / concentration:

Oral: 3 males/dose
Intravenous: 3 males/dose

Control animals:

no

Positive control reference chemical:

Not applicable

Details on dosing and sampling:

TOXICOKINETIC STUDY
- Tissues and body fluids sampled: Blood
- Blood samples (1 mL) were collected from a peripheral vessel into tubes containing K2EDTA as the anticoagulant.
- Time and frequency of sampling: Samples were collected from oral treated groups at approximately 0, 15, 30 min, 1, 2, 4, 8, and 24 hours post dose. Samples were collected from IV treated group at approximately 0, 2, 5, 10, 30 min, 1, 2, 4, and 8 hours post-dose.
- Sample collection, handling, storage and transfer was performed according to Charles River Laboratories Study 20033875 Protocol.
- The samples were stored on ice until centrifuged at 3500 rpm for 10 min at 5 ± 3 °C. The plasma samples were placed on dry ice prior to storage at approximately -20 °C. Samples were protected from direct sunlight during collection and processing. Plasma samples were transferred on dry ice to the Analytical Department at the test facility for analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) on October 1, 2012 and were stored at approximately -20 °C until analysis.

Statistics:

Statistical analyses were limited to descriptive statistics such as mean and standard deviation, if applicable. Because the data was computer-generated and rounded appropriately for inclusion in the report, the use of reported values to calculate subsequent parameters could, in some instances, yield minor variations from those listed in the tables.

Type:

absorption

Details on absorption:

Plasma Concentration
For oral administration, the mean concentration of S5353 in 24 hour samples is higher than that of 8 hour samples at dose 30 and 100 mg/kg. This reabsorption may be due to enterohepatic circulation.

Pharmacokinetic Evaluation
For intravenous administration, the mean values of terminal half-life for the test item in plasma were 0.56 ± 0.11 hours. For oral administration, the half-life mean values for the test item in plasma ranged from 1.92 ± 1.00 to 5.25 ± 3.29 hours. For intravenous administration, the mean values of terminal half-life for S2225 in plasma were 1.10 ± 0.04 hours. For oral administration, the half-life mean values for S2225 in plasma ranged from 2.22 ± 1.59 to 4.69 ± 2.08 hours. For intravenous administration, the mean values of terminal half-life for S5353 in plasma were 4.17 ± 0.19 hours. For oral administration, the half-life mean values for S5353 in plasma ranged from 4.42 ± 0.50 to 6.07 ± 1.82 hours. The test article in dog plasma was stable for at least 6 hours at room temperature during the bioanalytical method development. The exposure (AUClast) of S5353 in plasma is approximate 200 times that of S2225 and 800 times that of the test item. With oral administration, both mean AUClast and mean Cmax increased with dose up to 30 mg/kg in the animals.

Absolute Bioavailability
The absolute bioavailability (%F) ranged from 0.75 ± 0.48 to 1.26 ± 1.15. The low values of bioavailability may be due to the first-pass effect or losses in the gut lumen. Based on plasma concentration and dose proportionality, S5353 seems to be a good surrogate for oral bioavailability of the test item.

Animal Observations

For oral administration, all animals appeared healthy prior to dose and throughout the duration of the study. After intravenous administration, two dogs (1001 and 1002) exhibited moderate muscular tremors (shivering) accompanied by transient lethargy, elevated body temperature (102.5°F), and vocalization during and after dosing. All symptoms resolved within 15 minutes after dosing without veterinary intervention. As a result of the unexpected clinical observations noted above, the third animal in the group was not dosed. Also due to the unexpected clinical observations for Animals 1001 and 1002 during and immediately after dosing, the 2-minute blood sample was not collected from either animal.

 

Formulation Analysis

For intravenous administration, the test article formulation (5 mg/mL) was within the acceptance criteria of ±15% of the nominal concentration.

Conclusions:

The test article in dog plasma was stable for at least 6 hours at room temperature during the bioanalytical method development. The exposure (AUClast) of S5353 in plasma is approximate 200 times that of S2225 and 800 times that of the test item. With oral administration, both mean AUClast and mean Cmax increased with dose up to 30 mg/kg in male dogs. Based on plasma concentration and dose proportionality, S5353 seems to be a good surrogate for oral bioavailability of the test item.

Executive summary:

A study was conducted to determine the pharmacokinetic parameters of the test item and its hydrolysis metabolites (S2225 and S5353) in plasma following single oral administration as well as single intravenous administration in male beagle dogs.

 

For single intravenous administration, 2 animals per group were bolus injected with the test item at 1 mg/kg bw in PEG 400 solution. Blood samples were collected from a peripheral vessel at approximately 0, 5, 10, 30 min, 1, 2, 4, and 8 hours post-dose. For oral administration, 3 animals per group were single dosed with the test item at 10, 30, and 100 mg/kg bw in gelatin capsules by oral gavage. Blood samples were taken from a peripheral vessel at approximately 0, 15, 30 min, 1, 2, 4, 8, and 24 hours post-dose. Plasma samples were prepared by centrifugation, frozen and transferred to the Analytical Department at the test site for analysis of the test item, S2225, and S5353 by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The pharmacokinetic parameters were analyzed by non-compartmental methods using Phoenix WinNonlin software.

 

For intravenous administration, the mean values of terminal half-life for the test item in plasma were 0.56 ± 0.11 hours in male dogs. For oral administration, the half-life mean values for the test item in plasma ranged from 1.92 ± 1.00 to 5.25 ± 3.29 hours in the animals. For intravenous administration, the mean values of terminal half-life for S2225 in plasma were 1.10 ± 0.04 hours in the animals. For oral administration, the half-life mean values for S2225 in plasma ranged from 2.22 ± 1.59 to 4.69 ± 2.08 hours in the animals. For intravenous administration, the mean values of terminal half-life for S5353 in plasma were 4.17 ± 0.19 hours in the animals. For oral administration, the half-life mean values for S5353 in plasma ranged from 4.42 ± 0.50 to 6.07 ± 1.82 hours in the animals.

 

The test article, the test item, in dog plasma was stable for at least 6 hours at room temperature during the bioanalytical method development. The exposure (AUC_last) of S5353 in plasma is approximate 200 times that of S2225 and 800 times that of the test item. With oral administration, both mean AUC_last and mean C_max increased with dose up to 30 mg/kg bw in the animals. Based on plasma concentration and dose proportionality, S5353 seems to be a good surrogate for oral bioavailability of the test item.

Reference 3

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

metabolism

Qualifier:

no guideline available

Principles of method if other than guideline:

The purpose of this study was to evaluate the metabolism of test item in human, rat, dog, rabbit, and Gottingen pig hepatic microsomes in order to determine the similarity of the metabolic profile across species and to assess the suitability of the rat as a species for toxicology studies. Only Phase I metabolic reactions were monitored.

GLP compliance:

no

Radiolabelling:

no

Type:

metabolism

Metabolites identified:

yes

Details on metabolites:

The test item was more rapidly metabolized by either rat or pig than by the dog or human microsomes during the 60 minute incubation period. At the end of the incubation, roughly 7.67%, 3.15%, 1.00%, and 0.89% of test item was remaining for the dog, human, rat, and pig, respectively. A hydrolysis product, N-(thiophen-2-ylmethyl)-1H-pyrazol-3-amine (M179, S8229), was observed in microsomal incubations with and without NADPH (i.e., non-CYP450 dependent pathway). In the case of the rabbit microsomes, hydrolysis was rapid; less than 15% of test item remained at T = 0 min (i.e., time of NADPH addition). In all other species, greater than 98% of test item remained at T = 0 min. Since the test item was already mostly hydrolyzed in the rabbit microsome incubates at T = 0 min, the relative peak area for the hydrolysis product M179 compared to test item peak area at time 0 was well above 100%, and the area % relative to the test item area at T = 0 min for rabbit incubates corresponds to significantly lower metabolite concentration levels compared to the similar relative area % for the metabolites for the other species.

The other expected amide bond hydrolysis product, (p-tolyloxy)acetic acid (M166), was understandably not observed in positive ionization mode but was detected in negative ionization mode. It was assumed that equal molar amounts of M166 and M179 were initially generated by the hydrolysis reaction. M182A and M182B, the hydroxylation products of M166, were also observed in negative mode in rabbit and pig microsome incubates at apparently low abundance. Note that the relative peak areas for M166, M182A, and M182B were not listed since it is not meaningful to compare peak areas from different ionization modes.

A total of 27 metabolites were observed across all species. Because the test item is metabolized at different rates across species, metabolite profiles in human, rat, dog, and pig were compared at a time point where 3.15 - 9.01% of test item remained. The data from rabbit microsomes were not included in this comparison since the majority of the parent compound was already hydrolyzed at time 0. The amide bond hydrolysis product M179 is produced by all species in greater amounts than in human. The major Phase I metabolites produced in human microsomes result from the oxidative cleavage of the thienylmethyl group (M231), hydroxylation of the p-tolyloxy moiety (M343A), and from oxidation of the thiophene ring (M343D and M361). The metabolite profile in dog microsomes is nearly identical to that of the human. The rat does not produce significant quantities of the M231 and M361 metabolites, and also produces large amounts of M341A, a further oxidation product of M343D produced only in trace quantities in human microsomes, suggesting that the dog may be a more suitable model for toxicology studies with the test item. The abundance of the metabolic “hot spots” for the test item may be beneficial since its metabolic pathway of elimination does not have to rely on limited routes of biotransformation.
The proposed structures for the observed metabolites observed and their relative abundances are summarized in Tables 1 and 2.
The potential Phase I transformations of the test item are listed in Table 3 and the corresponding structures are listed in Table 4.

Refer "Attached background material" section for results of tables

Conclusions:

The test item was more rapidly metabolized by either rat or pig than by the dog or human microsomes during the 60 minute incubation period. A hydrolysis product, N-(thiophen-2-ylmethyl)-1H-pyrazol-3-amine (M179, S8229), was observed in microsomal incubations with and without NADPH. In the case of the rabbit microsomes, hydrolysis was rapid; less than 15% of the test item remained at T = 0 min (i.e., time of NADPH addition). In all other species, greater than 98% of the test item remained at T = 0 min.

Executive summary:

A study was conducted to evaluate the metabolism of the test item in human, rat, dog, rabbit, and Gottingen pig hepatic microsomes in order to determine the similarity of the metabolic profile across species and to assess the suitability of the rat as a species for toxicology studies. Only Phase I metabolic reactions were monitored. The test item (10 µM) was incubated with mixed gender, pooled liver microsomes (0.50 mg/mL) from human, rat, dog, and rabbit, or with pooled liver microsomes from male Gottingen pigs in the presence of NADPH at 37 °C for 10, 20, or 60 minutes prior to quenching the samples with acetonitrile. Control samples included time zero and 60 minute incubates without NADPH. Buspirone was tested in parallel with the test item to confirm the functionality of the microsomes. Samples were centrifuged to separate the precipitated microsomes from the supernatant containing the parent compound and its metabolites. The supernatant was analyzed by LC-MS/MS in order to evaluate the metabolism of the test item.

 

The test item was more rapidly metabolized by either rat or pig than by the dog or human microsomes during the 60 minute incubation period. At the end of the incubation, roughly 7.67%, 3.15%, 1.00%, and 0.89% of the test item was remaining for the dog, human, rat, and pig, respectively. A hydrolysis product, N-(thiophen-2-ylmethyl)-1H-pyrazol-3-amine (M179, S8229), was observed in microsomal incubations with and without NADPH (i.e., non-CYP450 dependent pathway). In the case of the rabbit microsomes, hydrolysis was rapid; less than 15% of the test item remained at T = 0 min (i.e., time of NADPH addition). In all other species, greater than 98% of the test item remained at T = 0 min.

 

A total of 27 metabolites were observed across all species. Because the test item is metabolized at different rates across species, metabolite profiles in human, rat, dog, and pig were compared at a time point where 3.15 - 9.01% of the test item remained. The amide bond hydrolysis product M179 is produced by all species in greater amounts than in human. The major Phase I metabolites produced in human microsomes result from the oxidative cleavage of the thienylmethyl group (M231), hydroxylation of thep-tolyloxy moiety (M343A), and from oxidation of the thiophene ring (M343D and M361). The metabolite profile in dog microsomes is nearly identical to that of the human. The rat does not produce significant quantities of the M231 and M361 metabolites, and also produces large amounts of M341A, a further oxidation product of M343D produced only in trace quantities in human microsomes, suggesting that the dog may be a more suitable model for toxicology studies with the test item. The abundance of the metabolic “hot spots” for the test item may be beneficial since its metabolic pathway of elimination does not have to rely on limited routes of biotransformation.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Objective of study:

absorption

Qualifier:

no guideline available

Principles of method if other than guideline:

The objective of this study was to determine the pharmacokinetic parameters of the test item and its hydrolysis metabolites (S2225 and S5353) in plasma following single oral administration as well as single intravenous administration in male and female Sprague-Dawley rats. This study also evaluated gender differences of the test item, S2225, and S5353 exposures in plasma.

GLP compliance:

no

Specific details on test material used for the study:

- Storage condition of test material: Stored at approximately 25 °C away from direct sunlight until the day of the experiment and/or analysis.

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Housing: Animals were individually housed.
- All animals were fasted overnight prior to treatment (total fasting time did not exceed 12 hours).
- On test day, rats were weighed and randomly assigned to study groups.
- Acclimation period: One week

Route of administration:

other: oral (gavage) and intravenous

Vehicle:

other: oral (1% methyl cellulose) and intravenous (PEG 400)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
To prepare 1% methyl cellulose (MC), the required amount of MC was weighed and added to approximately 10% of total volume of hot deionized water (100 °C) while stirring. Once MC particles were dispersed in the hot water, additional room temperature deionized water was added to form the 1% solution. All formulations were prepared one day before the experiment.
For each oral dose, vehicle (1% MC) was added to the required amount of the test item. Each suspension was stirred overnight. Immediately prior to administration of each dose, the suspension was stirred and then drawn up into the syringe.
For each IV dose, PEG 400 was added to the required amount of the test item and slowly heated until the compound was completely dissolved.

The test item was administered via single oral gavage or single intravenous bolus injection. The dose levels for the oral treated groups were 10, 30, and 100 mg/kg bw at a dose volume of ~2.5 mL/kg bw. The dose level for the intravenous groups was 1 mg/kg bw at a dose volume of ~0.25 mL/kg bw. Each dose was based on the body weight measured on the day of administration.

Duration and frequency of treatment / exposure:

Single dose

Dose / conc.:

1 mg/kg bw/day

Remarks:

intravenous

Dose / conc.:

10 mg/kg bw/day

Remarks:

oral

Dose / conc.:

30 mg/kg bw/day

Remarks:

oral

Dose / conc.:

100 mg/kg bw/day

Remarks:

oral

No. of animals per sex per dose / concentration:

Oral: 4 animals/sex/dose
Intravenous: 4 animals/sex/dose

Control animals:

no

Positive control reference chemical:

Not applicable

Details on dosing and sampling:

PHARMACOKINETIC STUDY
- Tissues and body fluids sampled: Blood
- Blood samples were collected from a jugular catheter into tubes containing K2EDTA as the anticoagulant.
- Time and frequency of sampling: Samples were collected from oral treated groups at approximately 0, 15, 30 min, 1, 2, 4, 8, and 24 hours post dose. Samples were collected from intravenous groups at approximately 0, 2, 5, 10, 30 min, 1, 2, 4, and 8 hours post-dose.
- The samples were stored on ice for no more than 15 min until centrifuged at 2 to 7 °C. The plasma samples were placed on dry ice prior to storage at approximately -70 °C. Samples were protected from direct sunlight during collection and processing. Samples were transferred to the Analytical Department at the test facility for analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS).

Statistics:

Statistical analyses were limited to descriptive statistics such as mean and standard deviation, if applicable. Because the data was computer-generated and rounded appropriately for inclusion in the report, the use of reported values to calculate subsequent parameters could, in some instances, yield minor variations from those listed in the tables.

Type:

absorption

Details on absorption:

Pharmacokinetic Evaluation
For intravenous administration, the mean values of terminal half-life for the test item in plasma were 0.30 ± 0.09 hours in female rats and 0.25 ± 0.01 hours in male rats. For oral administration, the half-life mean values for the test item in plasma ranged from 1.46 ± 0.94 to 2.91 ± 1.43 hours in female rats and 0.60 ± 0.28 to 3.02 ± 0.89 hours in male rats. For intravenous administration, the mean values of terminal half-life for S2225 in plasma were 0.82 ± 0.37 hours in female rats and 0.28 ± 0.04 hours in male rats. For oral administration, the half-life mean values for S2225 in plasma ranged from 1.02 ± 0.26 to 1.84 ± 0.44 hours in female rats and 1.12 ± 0.17 to 1.83 ± 0.25 hours in male rats. For intravenous administration, the mean values of terminal half-life for S5353 in plasma were 1.01 ± 0.73 hours in female rats and 0.86 ± 0.22 hours in male rats. For oral administration, the half-life mean values for S5353 in plasma ranged from 1.23 ± 0.28 to 2.85 ± 0.49 hours in female rats and 1.34 ± 0.35 to 3.29 ± 0.88 hours in male rats. With oral administration, both mean AUClast and mean Cmax on the test item and hydrolysis metabolites (S2225 and S5353) increased with doses in both female and male rats. The exposure (AUClast) of S5353 in plasma was approximately 10 times higher than S2225 and 1000 times higher than the test item.

Gender Difference
The exposures to the test item, S2225, and S5353 (Cmax and AUClast) in plasma were used for comparison of gender differences by ratio of female/male. For intravenous administration, the ratios of mean AUClast were 1.35 ± 0.48 for the test item, 1.43 ± 0.65 for S2225, and 1.83 ± 1.16 for S5353. The ratios of mean Cmax were 1.48 ± 0.58 for the test item, 0.93 ± 0.38 for S2225, and 1.69 ± 0.39 for S5353. For oral administration, the ratios of mean AUClast ranged from 3.14 ± 1.47 to 4.99 ± 4.22 for the test item, 1.51 ± 1.21 to 4.09 ± 3.08 for S2225, and 1.35 ± 0.56 to 1.75 ± 0.57 for S5353. The ratios of mean Cmax ranged from 2.54 ± 1.56 to 5.20 ± 6.39 for the test item, 1.75 ± 0.78 to 5.30 ± 4.18 for S2225, and 1.28 ± 0.83 to 2.01 ± 1.12 for S5353.

Absolute Bioavailability
The absolute bioavailability (%F) of the test item was not calculated due to the rapid hydrolysis of the test item in plasma. Based on plasma concentration and dose proportionality, S5353 seems to be a good surrogate for oral bioavailability of the test item.

Animal Observations

All animals appeared healthy prior to dose and throughout the duration of the study. Animal #25 (female, oral dose at 100 mg/kg) was not used for PK calculations due to contamination in the samples.

 

Formulation Analysis

All of the test article formulations (1, 3, and 10 mg/mL in 1% methyl cellulose) were within the acceptance criteria of ±20% of their nominal concentrations.

Conclusions:

The rapid hydrolysis of the test item to S2225 and S5353 in rat plasma was observed during the bioanalytical method development. The exposure (AUClast) of S5353 in plasma was approximately 10 times higher than S2225 and 1000 times higher than the test item. With oral administration, both mean AUClast and mean Cmax increased with dose in both female and male rats. Based on plasma concentration and dose proportionality, S5353 seems to be a good surrogate for oral bioavailability of the test item.

Executive summary:

A study was conducted to determine the pharmacokinetic parameters of the test item and its hydrolysis metabolites (S2225 and S5353) in plasma following single oral administration as well as single intravenous administration in male and female Sprague-Dawley rats. This study also evaluated gender differences of the test item, S2225, and S5353 exposures in plasma.

 

For single intravenous administration, 4 male and 4 female Sprague-Dawley rats per group were bolus injected with the test item at 1 mg/kg bw in PEG 400 solution. Blood samples were collected from a jugular catheter at approximately 0, 2, 5, 10, 30 min, 1, 2, 4, and 8 hours post-dose. For oral administration, 4 male and 4 female Sprague-Dawley rats per group were single dosed with the test item at 10, 30, and 100 mg/kg bw in 1% methyl cellulose by oral gavage. Blood samples were taken from a jugular catheter at approximately 0, 15, 30 min, 1, 2, 4, 8, and 24 hours post-dose. Plasma samples were prepared by centrifugation, frozen and transferred to the Analytical Department for analysis of the test item, S2225, and S5353 by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The pharmacokinetic parameters were analyzed by non-compartmental methods using Phoenix WinNonlin software.

 

For intravenous administration, the mean values of terminal half-life for the test item in plasma were 0.30 ± 0.09 hours in female rats and 0.25 ± 0.01 hours in male rats. For oral administration, the half-life mean values for the test item in plasma ranged from 1.46 ± 0.94 to 2.91 ± 1.43 hours in female rats and 0.60 ± 0.28 to 3.02 ± 0.89 hours in male rats. For intravenous administration, the mean values of terminal half-life for S2225 in plasma were 0.82 ± 0.37 hours in female rats and 0.28 ± 0.04 hours in male rats. For oral administration, the half-life mean values for S2225 in plasma ranged from 1.02 ± 0.26 to 1.84 ± 0.44 hours in female rats and 1.12 ± 0.17 to 1.83 ± 0.25 hours in male rats. For intravenous administration, the mean values of terminal half-life for S5353 in plasma were 1.01 ± 0.73 hours in female rats and 0.86 ± 0.22 hours in male rats. For oral administration, the half-life mean values for S5353 in plasma ranged from 1.23 ± 0.28 to 2.85 ± 0.49 hours in female rats and 1.34 ± 0.35 to 3.29 ± 0.88 hours in male rats.

 

The exposures to the test item, S2225, and S5353 (C_max and AUC_last) in plasma were used for comparison of gender differences by ratio of female/male. For intravenous administration, the ratios of mean AUC_last were 1.35 ± 0.48 for the test item, 1.43 ± 0.65 for S2225, and 1.83 ± 1.16 for S5353. The ratios of mean C_max were 1.48 ± 0.58 for the test item, 0.93 ± 0.38 for S2225, and 1.69 ± 0.39 for S5353. For oral administration, the ratios of mean AUC_last ranged from 3.14 ± 1.47 to 4.99 ± 4.22 for the test item, 1.51 ± 1.21 to 4.09 ± 3.08 for S2225, and 1.35 ± 0.56 to 1.75 ± 0.57 for S5353. The ratios of mean C_max ranged from 2.54 ± 1.56 to 5.20 ± 6.39 for the test item, 1.75 ± 0.78 to 5.30 ± 4.18 for S2225, and 1.28 ± 0.83 to 2.01 ± 1.12 for S5353.

 

The rapid hydrolysis of the test item to S2225 and S5353 in rat plasma was observed. The exposure (AUC_last) of S5353 in plasma was approximately 10 times higher than S2225 and 1000 times higher than the test item. With oral administration, both mean AUC_last and mean C_max increased with dose in both female and male rats. Based on plasma concentration and dose proportionality, S5353 seems to be a good surrogate for oral bioavailability of the test item.

Reference 5

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

metabolism

Qualifier:

no guideline available

Principles of method if other than guideline:

The objective of this study was to identify the metabolites of test item in plasma from the study of a single oral dose in male and female Sprague-Dawley rats as well as male beagle dogs.

GLP compliance:

no

Specific details on test material used for the study:

- Storage condition of test material: Stored as solids at approximately 25 °C away from direct sunlight until the day of the experiment and/or analysis

Radiolabelling:

no

Species:

other: rat and dog

Strain:

other: Sprague-Dawley rat and Beagle dog

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS - RAT
- Source: Charles River
- Housing: Animals were individually housed.
- All animals were fasted overnight prior to treatment (total fasting time did not exceed 12 hours).
- On test day, rats were weighed and randomly assigned to study groups.
- Acclimation period: One week

Route of administration:

other: oral (gavage) for rats; oral (capsules) for dogs

Vehicle:

methylcellulose

Remarks:

1% methyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
To prepare the vehicle, 1% methyl cellulose (MC), the required amount of MC was weighed and added to approximately 10% of total volume of hot deionized water (100 °C) while stirring. Once methyl cellulose particles were dispersed in the hot water, additional room temperature deionized water was added to form the 1% solution. All formulations were prepared one day before the experiment. For each oral dose, vehicle (1% MC) was added to the required amount of the test item. Each suspension was stirred overnight. Immediately prior to administration of each dose, the suspension was stirred and then drawn up into the syringe.

The test item was administered once for rats via oral gavage at a dose volume of ~10 mL/kg. Dose capsules for dogs were administered manually. Following each capsule dose, 10-20 mL of water was offered to each animal to assist in swallowing the capsule. All doses of the test item were based on the most recent body weight.

Duration and frequency of treatment / exposure:

Single dose

Dose / conc.:

100 mg/kg bw/day

No. of animals per sex per dose / concentration:

Rat - 4 animals/sex/dose
Dog - 3 males/dose

Control animals:

no

Positive control reference chemical:

Not applicable

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: Blood samples
- Blood samples from rats or dogs were collected from a jugular catheter or a peripheral vessel into tubes containing K2EDTA as the anticoagulant, respectively.
- Time and frequency of sampling: Samples were collected from rats or dogs at approximately 0, 15, 30 min, 1, 2, 4, 8, and 24 hours post dose.
- The samples were stored on ice for no more than one hour until centrifuged at 2 to 7 °C. The plasma samples were placed on dry ice prior to storage at approximately -70 °C. Samples were protected from direct sunlight during collection and processing.
- Method type(s) for identification: Analysis by liquid chromatography quadrupole time-of fly mass spectrometry (LC-QTOF).

Statistics:

No data

Type:

metabolism

Metabolites identified:

yes

Details on metabolites:

A total of 11 metabolites were observed in rat and dog plasma samples. In addition to the carboxylic acid M166 (S5353) and secondary amine M179 (S2225) amide bond hydrolysis products, five Phase I and two Phase II metabolites of the test item were observed in the rat plasma samples. The Phase I metabolic biotransformation of the test item in the rat involved oxidative cleavage of the thienylmethyl group to M231 (S9741), hydroxylation of the pyrazole moiety to M343B and M343C, hydroxylation and oxidation of the p-tolyloxy moiety to the corresponding alcohol M343A (S9742) and carboxylic acid M357D (S4068), hydrolysis of M343A to the carboxylic acid/alcohol M182 (S5956), and hydrolysis of M357D to the dicarboxylic acid M196 (S2636). Phase II metabolites consisted of the acyl glucuronide M533A (S8575) derived from carboxylic acid M357D and M519B derived from glucuronidation of hydroxylation M343C. Based on mass spec peak areas, metabolites M166, M179, M357D, M343C, and M533A were present at higher concentrations than the parent compound the test item throughout the entire 24 hour collection period. Alcohol M343A appears to be rapidly converted to acid M357D, which in turn, is converted to acyl glucuronide M533A in the rat.

With the exception of oxidative metabolites M343C, M231, M182, and dicarboxylic acid M196, the same Phase I metabolites were also observed in dog plasma. In addition to the acyl glucuronide metabolite M533A, a second Phase II metabolite M519A (S6432) derived from glucuronidation of alcohol M343A, was also observed in the dog. Based on mass spec peak areas, carboxylic acids M166 and M357D, and glucuronides M519A and M533A, were the major metabolites observed in dog and were present at higher concentrations than the parent compound the test item throughout the entire 24 hour collection period. As was observed in the case of the rat, alcohol M343A appears to be a transient intermediate which is rapidly converted to metabolites M357D, M519A, and M533A in the dog.

In general, the plasma concentrations of the Phase I metabolites were higher in the rat than in the dog. The identities of nine of the rat/dog metabolites were confirmed by direct comparison to synthetic standards.

Animal Observations

All animals appeared healthy prior to dose and throughout the duration of the study.

 

Formulation Analysis

The test articles in suspension formulations (10 mg/mL in 1% methyl cellulose) for rats were within the acceptance criteria of ±20% of their nominal concentrations.

In Vivo Metabolism of the test item

Agilent Masshunter Metabolite ID software (Revision B.04.00) was used to identify metabolite candidates of the test item. Both positive and negative charged ions were used to analyze for potential metabolites with this software. Based on biotransformations and temporal profile of peak area, eleven metabolites of the test item in the rat and dog plasma at the 1 hour time point were selected to report with a peak area above 3000 which is approximate limit of detection. Eight metabolites were detected from positive charged ions and three metabolites were observed from negative charged ions. The proposed chemical structures for metabolites are shown in Figure 1.

 

Refer "Attached background material" section for results of tables and figures

Conclusions:

Seven Phase I and two Phase II metabolites of the test item were observed in the rat samples. Four Phase I and two Phase II metabolites of the test item were observed in the dog samples. Eight metabolites were confirmed by synthetic standards. In general, the plasma concentrations of these metabolites were higher in the rat than in the dog. Based on both the pharmacokinetic and in vivo metabolism data in both rat and dog, there does not appear to be support for the dog being a more appropriate model for human toxicity over the rat in the case of the test item.

Executive summary:

A study was conducted to identify the metabolites of the test item in plasma from the study of a single oral dose in male and female Sprague-Dawley rats as well as male beagle dogs.

 

For the determination of metabolites in plasma, 4 male and 4 female Sprague-Dawley rats, or 3 male dogs per group were administered the test item at 100 mg/kg in 1% methylcellulose by oral gavage (rats) or by gelatin capsule (dogs). Blood samples were taken at approximately 0, 15, 30 min, 1, 2, 4, 8, and 24 hours post dose. Plasma samples were analyzed for the test item and metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Agilent 6550 iFunnel QTOF equipped with a 1290 UPLC system. Test article formulations prepared for rats were analyzed for concentration before and after dosing by HPLC-UV.

 

A total of 11 metabolites were observed in rat and dog plasma samples. In addition to the carboxylic acid M166 (S5353) and secondary amine M179 (S2225) amide bond hydrolysis products, five Phase I and two Phase II metabolites of the test item were observed in the rat plasma samples. The Phase I metabolic biotransformation of the test item in the rat involved oxidative cleavage of the thienylmethyl group to M231 (S9741), hydroxylation of the pyrazole moiety to M343B and M343C, hydroxylation and oxidation of thep-tolyloxy moiety to the corresponding alcohol M343A (S9742) and carboxylic acid M357D (S4068), hydrolysis of M343A to the carboxylic acid/alcohol M182 (S5956), and hydrolysis of M357D to the dicarboxylic acid M196 (S2636). Phase II metabolites consisted of the acyl glucuronide M533A (S8575) derived from carboxylic acid M357D and M519B derived from glucuronidation of hydroxylation M343C. Based on mass spec peak areas, metabolites M166, M179, M357D, M343C, and M533A were present at higher concentrations than the parent compound the test item throughout the entire 24 hour collection period. Alcohol M343A appears to be rapidly converted to acid M357D, which in turn, is converted to acyl glucuronide M533A in the rat.

 

With the exception of oxidative metabolites M343C, M231, M182, and dicarboxylic acid M196, the same Phase I metabolites were also observed in dog plasma. In addition to the acyl glucuronide metabolite M533A, a second Phase II metabolite M519A (S6432) derived from glucuronidation of alcohol M343A, was also observed in the dog. Based on mass spec peak areas, carboxylic acids M166 and M357D, and glucuronides M519A and M533A, were the major metabolites observed in dog and were present at higher concentrations than the parent compound the test item throughout the entire 24 hour collection period. As was observed in the case of the rat, alcohol M343A appears to be a transient intermediate which is rapidly converted to metabolites M357D, M519A, and M533A in the dog.

 

In general, the plasma concentrations of the Phase I metabolites were higher in the rat than in the dog. The identities of nine of the rat/dog metabolites were confirmed by direct comparison to synthetic standards.

 

Seven Phase I and two Phase II metabolites of the test item were observed in the rat samples. Four Phase I and two Phase II metabolites of the test item were observed in the dog samples. Eight metabolites were confirmed by synthetic standards. In general, the plasma concentrations of these metabolites were higher in the rat than in the dog. Based on both the pharmacokinetic and in vivo metabolism data in both rat and dog, there does not appear to be support for the dog being a more appropriate model for human toxicity over the rat in the case of the test item.

Description of key information

The available evidence suggests that the substance is bioavailable via the oral, dermal and inhalation route. The substance is expected to be mainly excreted in urine.The substance has low potential to bioaccumulate.

Additional information

In accordance with the section 8.1.1 of Annex VIII of Regulation (EC) No 1907/2006 (REACH), the toxicokinetic profile of the substance (i.e. absorption, distribution, metabolism and elimination) was derived from the relevant available information collated in the dossier. The physical chemical characteristics of the registered substance, the results obtained from acute and repeated-dose toxicity studies as well as information gained from genotoxicity assays were used to predict its toxicokinetic behaviour.

 

Physical-chemical properties:

The substance is a mono-constituent, having a relatively low molecular weight of 327.4 g/mol. The substance is a slightly water-soluble solid/powder (13.2 mg/L) and is moderately lipophilic based on the octanol/water partition coefficient (Log Kow = 2.4). The substance has very low volatility according to its vapour pressure (0.054 Pa at 20°C).

 

Absorption:

Oral/GI absorption

The physical chemical characteristics described above suggest that the registered substance is of adequate molecular size to participate in endogenous absorption mechanisms within the mammalian gastrointestinal tract. Being lipophilic, the registered substance may be expected to cross gastrointestinal epithelial barriers where the absorption may be potentiated by the ability of the substance to dissolve into gastro-intestinal fluids and hence make contact with the mucosal surface.

No effects were observed in acute oral or repeated dose toxicity studies. Therefore the hypotheses of oral absorption is not supported by oral systemic effects.

In light of these data, and the lack of specific information, the substance was assumed to be 100% bioavailable by oral route for the purpose of human health risk assessment.

 

Dermal absorption

Regarding dermal absorption, the registered substance being moderately lipophilic (log Kow = 2.4) dermal absorption is expected to be high. However it is is assumed that the dermal uptake is limited by the low water solubility of the substance.

In light of these data, and the lack of specific information on the substance, a dermal absorption of 100% was conservatively assumed for the purposes of human health risk assessment.

 

Respiratory absorption

The potential for inhalation toxicity was not evaluated in vivo.

The vapour pressure of the substance (0.054 Pa at 20°C) indicated an absence of volatility. However the substance is a powder. No data are available on particle size and particle size distribution. However, a fraction of this substance could be inhaled as dust particles. Therefore exposure by inhalation could be anticipated. However, the substance is currently neither handled nor placed on the market under the solid form (powder). Moreover, the powder is manufactured in closed conditions, with immediate dilution in solvent. Thus, at ambient temperature, no respiratory absorption is expected under normal use and handling of the substance.

However, when used as a vapour or as mist (droplet aerosol), the substance is expected to be directly absorbed across the respiratory tract epithelium by passive diffusion.

In light of these data, and the lack of specific information on respiratory absorption, the substance was conservatively assumed to be 100% bioavailable by inhalation for the purposes of human health risk assessment.

 

Distribution:

Any material that is absorbed will be distributed via the blood to the liver, and other organs and tissues.

Systemic distribution of the substance can be predicted from its physical chemical characteristics.

Considering that the substance is slightly water soluble, it is suggested that, upon systemic absorption by oral or dermal route, or by inhalation, the substance may be transported through the circulatory system in association with a carrier molecule such as a lipoprotein or other macromolecule.

Based on its moderate lipophilic character, the substance may readily cross cellular barriers or may be distributed into fatty tissues with a low potential to accumulate (Log Kow < 4).

 

Metabolism:

Specific data on metabolism of the registered substance are available [1]: The in vitro metabolism of the substance was studied using rat, dog, rabbit, Gottingen pig, and human liver microsomes. The ex-vivo plasma stability of the test item was evaluated in rat, dog, rabbit, monkey, Gottingen pig, and human plasma. The in vivo metabolism and PK of the test item was studied in both rats and dogs. The PK of both the substance and its secondary amine hydrolysis product M179 was also determined in mice.

The substance undergoes rapid oxidative metabolism in vitro, and in rat and dog pharmacokinetic studies is rapidly converted to its component carboxylic acid and secondary amine [1].

On oral administration of the substance to either mouse, rat, or dog, the exposure (AUC) to the carboxylic acid hydrolysis product M166 was significantly higher than to either the parent amide or to the secondary amine hydrolysis product M179. This appears to be particularly true in the dog and in the mouse, where only very low levels of M179 were seen in plasma. The observation that amine M179 is not produced in proportion to carboxylic acid M166 suggests that either M179 is undergoing further metabolism, or that the amine moiety of the parent substance is being metabolized prior to cleavage of the amide bond. However, the plasma half-life of M179 in the dog is not significantly different from that of M166 suggesting that the latter may be the case. This is further supported by the observation that oral administration of M179 hydrochloride salt to mice results in significantly higher exposure of M179 in plasma (158-fold) than an equimolar dose of the parent substance, and also indicates that hydrolysis of the parent substance is not occurring to a significant extent in the intestinal lumen [1].

In addition to the carboxylic acid M166 and secondary amine M179 amide bond hydrolysis products, seven Phase I and two Phase II metabolites of the registered substance were observed in the rat plasma samples. A third Phase II metabolite, glucuronide M519A, was only seen in dog plasma. None of the microsomal metabolites involving oxidation of the thiophene ring were seen in either the rat or dog plasma samples at any time point. Most of the remaining human microsomal Phase I metabolites were present in both rat and dog plasma and tended to be present in higher concentrations in the rat than in the dog. Based both the pharmacokinetic and in vivo metabolism data in both rat and dog, the rat was viewed as a suitable species for the evaluation of the in vivo toxicology of registered substance [1].

 

Excretion:

The registered substance, having a molecular weight higher but close to 300 g/mol (ie. 327.4 g/mol), is expected to be excreted in urine and may be excreted in bile. Any substance that is not absorbed from the gastro-intestinal tract, following oral ingestion, will be excreted in the faeces..

 

Hydrolysis:

The substance was rapidly converted to its component carboxylic acid M166 and secondary amine M179 (N-(2-thienylmethyl)-1H-pyrazol-3-amine) on both intravenous and oral administration in mouse, rat, and dog [1].

 

Reference:

[1]Toxicological evaluation of a novel cooling compound: 2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-(2-thienylmethyl)acetamide. Donald S. Karanewsky, Amy J. Arthur∗, Hanghui Liu, Bert Chi, Stacy MarkisonSenomyx, Inc., 4767 Nexus Centre Drive, San Diego, CA 92121, USA. Toxicology Reports 2 (2015) 1291–1309